CDK2 regulates collapsed replication fork repair in CCNE1-amplified ovarian cancer cells via homologous recombination

Abstract CCNE1 amplification is a common alteration in high-grade serous ovarian cancer and occurs in 15–20% of these tumors. These amplifications are mutually exclusive with homologous recombination deficiency, and, as they have intact homologous recombination, are intrinsically resistant to poly (ADP-ribose) polymerase inhibitors or chemotherapy agents. Understanding the molecular mechanisms that lead to this mutual exclusivity may reveal therapeutic vulnerabilities that could be leveraged in the clinic in this still underserved patient population. Here, we demonstrate that CCNE1-amplified high-grade serous ovarian cancer cells rely on homologous recombination to repair collapsed replication forks. Cyclin-dependent kinase 2, the canonical partner of cyclin E1, uniquely regulates homologous recombination in this genetic context, and as such cyclin-dependent kinase 2 inhibition synergizes with DNA damaging agents in vitro and in vivo. We demonstrate that combining a selective cyclin-dependent kinase 2 inhibitor with a DNA damaging agent could be a powerful tool in the clinic for high-grade serous ovarian cancer.


INTRODUCTION
Ovarian cancer is the leading cause of death from gynecologic cancer, and the fifth leading cause of cancer-related deaths in women in the United States; in 2020, approximately 200 000 w omen w orldwide died from ovarian cancer ( 1 ). This disease generally has a poor prognosis, with a 5-year survival rate of only one-third of patients ( 1 ). Patients with homologous recombination deficient (HRD) ovarian cancer, such as those with mutations in BRCA1 / 2 , have showed clinical benefit from poly (ADP-ribose) polymerase (PARP) inhibitors, due to the synthetic lethal rela tionship between alterna ti v e non-homologous end joining (NHEJ), a pathway controlled in part by PARP, and homologous recombination (HR) ( 2 , 3 ). It has been previously reported tha t CCNE1 -amplifica tion, which occurs in 15-20% of high-grade serous ovarian cancer (HGSOC) patient tumors, generally does not co-occur with loss of function mutations of HR genes ( 4 , 5 ). It is belie v ed that because CCNE1 -amplified tumors have intact HR, they are resistant to PARP inhibitors and platinum agents, which is the standard of care for HGSOC ( 6 ). HGSOC patients with CCNE1-amplified tumors, ther efor e, have limited therapeutic options and r epr esent a high unmet medical need.
Cyclin-dependent kinase 2 (CDK2) is the canonical partner of cyclin E1, and this complex controls entry into the S-phase ( 7 ) via phosphorylation of the retinoblastoma protein 1 (RB1) to release E2F and allow for transcription of genes that will dri v e the cell cycle ( 8 ). Aberrant activation of the RB1-E2F pathway, such as through ov ere xpression of CCNE1 , increases double-stranded break (DSB) formation, leading to genomic instability ( 9 , 10 ). Specifically, CCNE1 ov ere xpression induces premature S-phase entry ( 11 ), incr easing r eplication fork stalling and collapse via insufficient nucleotide pools ( 12 ), or replicationtranscription collisions stemming from inappropriate origin firing ( 13 ). Although some studies have demonstrated a synthetic lethality between CCNE1 amplification and HRD, such as through genetic suppression of BRCA1 / 2 ( 14 ), other large-scale screens have not supported this relationship ( 15 , 16 ). The discrepancy in these results may stem from the experimental conditions (e.g. clonogenic assays versus logarithmic growth). More investigation is required to understand the molecular mechanism(s) underlying the mutual e xclusi vity of these two genomic e v ents.
In addition to their role in cell cycle progression, the cyclin-dependent kinases (CDKs) are known regulators of HR in eukaryotic cells. Cyclin-dependent kinase 1 (CDK1) and CDK2 have been implicated in HR in human cells via substra te phosphoryla tion during each step of recombination, but with conflicting evidence to support whether cells are dependent on CDK1, CDK2 or both for this pathway (17)(18)(19)(20)(21)(22)(23)(24). These studies are in part confounded by the use of non-selecti v e CDK inhibitors, making it difficult to ascribe specific functions to particular CDK family members. Moreover, the role of CDKs in DNA repair has not been well studied in a context where the core cell cycle machinery is d ysregula ted, as is the case in CCNE1 -amplified cells.
Here, we provide evidence for a mechanism that could explain the mutual e xclusi vity of CCNE1 amplification and HRD in HGSOC. We demonstrate that CCNE1 -amplified HGSOC cells rely on HR to resume replication following induced fork collapse. We detected high levels of CDK2 and cyclin E1 at the site of stalled replication forks only in CCNE1 -amplified cells, which places CDK2-cyclin E1 directly at barriers to replication. Lastly, we provide evidence that CDK2 is r equir ed for both signaling after replication fork stalling and HR in CCNE1 -amplfied cells, but is dispensable in CCNE1 non-amplified cells. Inhibition of CDK2 at low doses sensitizes CCNE1 -amplified cells to DNA-dama ging a gents both in vitro and in viv o . With the advancement of CDK2 inhibitors into the clinic, these studies suggest that the combination of CDK2 inhibition with DN A-damaging chemothera py or PARP inhibitors may provide clinical benefit in HGSOC patients with CCNE1amplified tumors.

Cell lines and culture
OVCAR-3 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 20% fetal bovine serum. hTERT-RPE1 cells were maintained in Dulbecco's Modified Eagle Medium: F12 (DMEM: F12) supplemented with 10% fetal bovine serum and 0.01 mg / ml hygromycin B. A549 cells were maintained in F-12K Medium supplemented with 10% fetal bovine serum. COV644 and COV318 cells were maintained in DMEM supplemented with 10% fetal bovine serum. FUOV1 cells were maintained in Ham's F12 + DMEM at 1:1, and supplemented with 10% fetal bovine serum. MCF-7 cells were maintained in Eagle's Minimum Essential Medium (EMEM), and supplemented with 0.01 mg / ml human recombinant insulin and 10% fetal bovine serum. OVCAR-3, hTERT-RPE1, A549, and MCF-7 cells were purchased from the American Type Culture Collection. COV644 and COV318 were purchased from Sigma Aldrich. FUOV1 cells were purchased from DSMZ. Cell line identity was confirmed with short tandem repeat (STR) testing (Charles Ri v er). All cells were maintained in a humidified incubator at 37 • C with 5% CO 2 .

Pr oliferation assa y
O VCAR-3, CO V644, or MCF-7 cells were seeded at 1000 cells / well in 384-well black, clear bottom plates and allowed to adhere overnight at 37 • C / 5% CO 2 . The following day, cells were treated with compound in a 10-point dose response, 1:4 dilutions. After 5 days of incubation, CyQuant Direct Proliferation Assay was performed according to the manufacturer's instructions. Data were processed with GraphPad Prism using a four-parameter fit to calculate IC 50 .

AlphaLISA assays
For the RB1 phosphorylation (T821 / 826) AlphaLISA, OVCAR-3 cells were seeded in serum-free DMEM in a 96well plate (25 000 cells / well) the day before the assay. After overnight incubation, media wer e r eplaced with DMEM containing 10% FBS, and cells were treated with a dose response of compound (1:4 dilutions) for 18 h. pRb Al-phaLISA was performed according to the manufacturer's instructions. Plates were read on EnVision plate reader (Perkin Elmer). Data were processed with GraphPad Prism using a four-parameter fit to calculate IC 50 .
For the Lamin phosphorylation (S22) AlphaLISA, OVCAR-3 or COV644 cells were seeded in DMEM containing 10% FBS in 384-well plates (3000 cells / well for OVCAR-3; 12 500 cells / well for COV644). After overnight incubation, cells wer e tr eated with a dose response of compound (1:4 dilutions) for 2 h. Media was aspirated from the plate, and cells were lysed in 10 l of 1 × AlphaLISA lysis buffer (Perkin Elmer Cat# AL003C) and placed on a plate shaker for 15 min. Anti-total lamin (SC-7292) was diluted to 0.2 nM, and anti-phospho Lamin (CST# 13448) was diluted to 0.3 nM in 1 × immunoassay buffer (Perkin Elmer Cat# AL00F). 10 l of each antibody was added to the plate and incubated for 1 h at RT. Anti-rabbit acceptor beads (Perkin Elmer Cat# AL104R) were prepared at 5 × and added to the plate (10 l / well) and incubated for 1 h at RT. Anti-mouse donor beads (Perkin Elmer Cat# AS104R) wer e pr epar ed at 5 × and added to the plate (10 l / well) and incubated overnight in the dark. Plates wer e r ead on EnVision plate reader (Perkin Elmer). Data were processed with GraphPad Prism using a four-parameter fit to calculate IC 50 .

Lentivir al tr ansduction / stable cell line gener ation
Lenti virus encoding inducib le non-targeting control shRN A, CDK2 shRN A or CDK1 shRN A in the SMARTv ector inducib le lenti viral shRNA v ector under the EF1 alpha promoter with puromycin resistance wer e pur chased from Horizon Discov ery. Stab le OVCAR-3, FUO V1, CO V318, hTERT-RPE1, A549 and COV644 cells were generated by exposing cells to lentivirus at a multiplicity of infection of 1:1 overnight in the presence of 5 g / ml polybrene. After overnight incubation, media was replaced and cells were allowed to recover for 48 h. Cells were passaged into selection media (Puromycin -O VCAR-3: 2 g / ml, FUO V1: 0.5 g / ml, CO V318: 5 g / ml, hTERT-RPE1: 10 g / ml, A549 and COV644: 1 g / ml) for 2 weeks before experiments were conducted.
CRISPR / cas9 recombination assay pBluescript SK II + and LentiCRISPR v2 plasmids were purchased from Genscript. LentiCRISPR v2 plasmids expressing single guide (sgRNA) against ACTB or scrambled sgRNA were constructed according to the depositor's instructions. To construct HR donor vectors against ACTB, sequences from −1554 to +1527 relati v e to the stop codon of ACTB plus 3 × FLAG directly 5' from the stop codon were synthesized and cloned into pBluescript SK II using restriction digest and ligation. Compounds were added to cells on the day of plasmid transfection. sgRNA / Cas9 and the donor plasmid were prepared in a 1:2 molar ratio; 1 g of total DNA / well was transfected onto cells using Lipofectamine 2000 (Thermo Fisher Scientific Cat#11668019). Cells were harvested for Western Blot analysis 72 h later.

In situ proximity ligation assay for EdU (double stranded DNA)-protein interaction
Cells were seeded onto microscope chamber slides the day before the experiment in DMEM supplemented with 10% FBS. On the day of the experiment, cells were incubated with 100 M EdU for 10 min and tr eated with hydroxyur ea (2 mM) for 2 h. After treatment, cells were pre-extracted in CSK-100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2, 10 mM Pipes pH 6.8, 1 mM EGTA, 0.2% Triton X-100, 1 × antiproteases) for 5 min on ice under gentle agitation and fixed with 4% PFA / PBS for 20 min at room temperature (RT). Cells were permeabilized in 0.2% Triton X-100 / PBS for 10 min at RT. Cells were incubated for 30 min at RT with Click-iT reaction cocktail (Thermo Fisher, Cat#C10269) to attach a biotin azide (ThermoFisher, Cat# B10184) to EdU. Next, cells were incubated with PLA blocking buffer (Sigma Aldrich) for 1 h at 37 • C in a humidified chamber before addition of primary antibodies: rabbit anti-CCNE1 (Sigma-Aldrich, 1:250); mouse anti-CDK2 (Santa Cruz,1:50); mouse anti-CCNA2 (Santa Cruz, 1:800); rabbit anti-biotin (CST,1:200), and mouse antibiotin (Sigma, 1:100). The negati v e control used only one primary antibody. Slides were incubated with secondary antibodies conjugated with PLA probes MINUS (anti-rabbit) and PLUS (anti-mouse) (Millipore Sigma). The incubation with all antibodies was done in a humidified chamber for 1 h a t 37 • C . PLA probes MINUS and PLUS were ligated using two connecting oligonucleotides to form a template for rolling-cycle amplification. Following amplification, the products were hybridized with red fluorescence-labeled oligonucleotide. Samples were mounted in Duolink In Situ mounting medium with DAPI (blue) (Sigma-Aldrich, Cat# DUO82040). Images were acquired using the Nikon Ti inverted, spinning disk confocal microscope 'Spinster' in the MicRoN (Microscop y Resour ces on the North Quad) cor e at Harvard Medical School. PLA spots per nuclei were quantified in each plane of the Z-stack using arivis Vi-sion4D analysis software on the MicRoN Big Dipper Image Analysis work station. Images used in manuscript r epr esent maximum projects of a Z-stack from ImageJ FIJI.

NanoBRET target engagement assays
HEK293 cells were prepared to a final concentration of 100 000 cells / ml in 1% fetal bovine serum (FBS) Opti-MEM. Transfection mixtures were prepared with 1 g of plasmid encoding NanoLuc-fused CDK and 9 g of plasmid encoding cyclin, diluted in 1 ml Opti-MEM. FuGENE HD (Promega) was added to transfection mixture at a ratio of 30 l / 1 ml. After liposome formation, the lipid:DNA complex was added at a 1:20 ratio to HEK293 cells in suspension. Cells were plated in 384-well tissue culture plates and incubated overnight (20-30 h) at 37 • C / 5% CO 2 . Compounds wer e pr epar ed from DMSO stock solutions and added to cell plates with a final top concentration of 10 000 nM. NanoBRET tracer solution was pr epar ed from 100X tracer solution. For CDK1 / cyclin B1, CDK9 / cyclin T1, and CDK2 / cyclin E1 assays, tracer K10 was used at a final concentration of 0.5 M, 0.250 M, and 0.125 M, respecti v ely. For CDK4 / cy clin D1, and CDK6 / cy clin D3, tracer K7 was used at a final concentration of 0.0625 M. For CDK7 / cyclin H1, tracer K7 was used at a final concentration of 0.5 M. The plate was incubated at 37 • C / 5% CO 2 for 2 h. NanoBRET substrate solution was pr epar ed 15 min prior to the assay endpoint using serum-free Op-tiMEM. After the incubation was complete, NanoBRET substrate solution was added to the treated cell plate. The plate was then shaken and read using the Perkin Elmer Envision plate reader. The donor emission was measured at 450 nm, followed by acceptor emission at 610 nm. BRET r atio was gener ated by dividing the acceptor emission signal by the donor emission signal per sample, and converted to milliBRET by m ultipl ying each raw value by 1000. Data were normalized to no drug (100% signal) and no tracer (0% signal), and fit using GraphPad Prism v8.0.2.

Cell cycle profiling
Reagents for EdU incorporation were included in the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific Cat#C10420). Cells were treated with compound for 22 h prior to EdU pulsing. EdU was added to cells in culture for a final concentration of 10 M, and incubated at 37 • C in 5% CO 2 for 2 h prior to collection and pelleting. Cells were incubated in the dark for 15 min with 100 l of Click-iT fixati v e, washed with 1% BSA PBS, and incubated for 15 min with 100 l 1x Click-iT saponinbased permeabilization agent, pr epar ed by 1:10 dilution of stock solution in PBS supplemented with 1% BSA. The Click-iT reaction cocktail was prepared following manufacturer's instructions and added to cells for 30 min at room temperature in the dark. Cells were washed with 1% BSA. FxCycle Violet (Thermo Fisher Scientific Cat#F10347) was pr epar ed in 1 × saponin-based permeabilization reagent at a 1:2000 dilution, and added to cells for 30 min at room temperature in the dark. FACS analysis was performed using LSR Fortessa (BD Biosciences) and analyzed using FlowJo.

Synergy analysis
OVCAR-3 or COV644 cells were seeded in 96-well plates in complete media. The next day, BLU1851 was added in a 10point dose response. After 24 h of incubation, etoposide was added in a fiv e-point dose response. The following day, compound containing media was aspirated, cells washed, and complete media added back. CyQuant was used 3 days later to determine cellular proliferation. HSA scor es wer e determined using Synergy Finger ( https://synergyfinder.org/ ).

In vivo tumor growth inhibition, pharmacokinetics, and pharmacodynamics
Female NOD-SCID mice were purchased from Shanghai Lingchang BioTech Co., Ltd at 6-8 weeks old. Mice were housed in solid bottom polycarbonate ventilated cages at a temperature of 20-26 • C in 40-70% humidity. Mice were kept on a 12-h light, Twelv e-hour dar k cy cle with regularly changed 100% filtered air. Municipal water and rodent diet were provided ad libitum. The general health of the animals was monitored thr ough gr oss observa tion, regular bod y weight measurement, and monitoring of food and water intake. All procedur es r elated to animal handling, car e, and tr eatment wer e performed according to the protocol approved by the Institutional Animal Care and Use Committee of Shanghai Chempartner following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
OVCAR-3 cells were passaged through mice once, and a cell line was rederi v ed from the tumor, named 'T2A.' OVCAR-3 T2A cells wer e cultur ed in RPMI 1640 media supplemented with 20% FBS and 0.01 mg / ml insulin. To establish the OVCAR-3 xenograft model, 6 × 10 6 cells per mouse were implanted subcutaneously into the right hind flank of NOD-SCID female mice. Cells were suspended in 50% Matrigel and 50% RPMI 1640 media serum free media prior to implantation.
In OVCAR-3 tumor growth inhibition studies, animals were randomized and treatment began when tumors reached ∼200 mm 3 in size ( n = at least eight / group). BLU2256 was pr epar ed in 20% in 100 mM citrate buffer, pH 3, and dosed orally (PO) as a suspension once daily (QD) at 3 mg / kg, or BID at 5 mg / kg. Etoposide was pr epar ed in 5% DMSO + 40% PEG300 + 5%Tween 80 + 50%dd water and dosed intra peritoneall y QD at 1.25 mg / kg for 3 days for each week of the study. Olaparib was pr epar ed in 10% HP-ß-CD in water and dosed intra peritoneall y QD at 50 mg / kg. Tumor sizes and body weight were measured twice w eekly. Tumor sizes w er e measur ed in two dimensions using a caliper, and the volume (mm 3 ) was calculated using the formula: V = 0.5 a x b 2 where a and b are the long and short diameters of the tumor, respecti v ely.

TCGA and DepMap data sources
We used publicly available data of patients with ovarian cancer from the TCGA to determine mutual e xclusi vity of CCNE1 amplification and mutations in HR-related genes. To interrogate features with potential relationships to HR, we retrie v ed data from the DepMap 21Q4 public release (DepMap, Broad, 2021) of annotated cell lines from the CCLE. Specifically, fusion and copy number data were used to assess the landscape of gross chromosomal rearrangements across cancer types and tumor mutation burden estimated from mutation data and aneuploidy scores deri v ed from ABSOLUTE copy number profiles were used to characterize differences in CCNE1 -amplified versus nonamplified cell lines.

Statistical analysis of mutual e x clusivity in cancer patients and fusion events in cancer cell lines
Mutual e xclusi vity of CCNE1 amplification and HRrelated genes was analyzed in patients with ovarian cancer with mutation and copy number data provided by TCGA. CCNE1 -amplified tumors had GISTIC-called amplifications w hile HR-m utated tumors had either missense, inframe, or trunca tion muta tions identified by whole exome sequencing ( 25 ). The Fishers' exact test was used to determine statistical significance ( P ≤ 0.05), while odds ratios were calculated to assess the magnitude and direction of effect.
Fusions identified in DepMap 21Q4 release were called from RNA-sequencing data using the STAR-Fusion pipeline version 1.6.0 and filtered as described in DepMap documentation ( 26 ). To create a more robust call set, we further filtered out fusions with fusion fragments per million < 0.1 or a spanning fragment count of 0 with no long double anchor support. Copy number values greater than thr ee wer e called as amplified, assuming diploid y. Dif ferences in fusion distribution, tumor mutation burden, and aneuploidy were tested using the Wilco x on rank-sum test ( P ≤ 0.05). Differences in the proportion of cell lines were tested using the Fishers' exact test ( P ≤ 0.05).
All statistical analyses were performed in R 3.6.3.

CCNE1-amplified o v arian cancers have a high incidence of chromosomal fusion events
Co-occurrence of CCNE1 amplification with RB1 loss and soma tic muta tions in genes involved in HR, Fanconi anemia (FA), and DNA repair was assessed using patient data from The Cancer Genome Atlas (TCGA). In agreement with previous analyses, CCNE1 amplification has a tendency toward mutual exclusivity with RB1 loss and putative dri v er mutations in the HR and FA pathways ( 4 ) (Figure  1 A, Supplementary Table S1). We also analyzed PIK3CA , a frequently amplified oncogene in ovarian cancer, and observed that PIK3CA and CCNE1 amplification are not mutually e xclusi v e (Supplementary Tab le S1). Because CCNE1 amplification is not generally mutually e xclusi v e with other oncogenic alterations, such as PIK3CA amplification, the mutual e xclusi vity between CCNE1 amplification and HR may be governed by specific molecular mechanisms. Because CCNE1 amplification induces oncogenic stress and DSBs that can lead to allelic imbalances ( 10 , 27 ), we assessed gr oss chr omosomal rearrangements (GCRs) in CCNE1 -amplified cell lines versus non-amplified cell lines using data from the Cancer Cell Line Encyclopedia (CCLE). Across all indications, we found that CCNE1amplified lines are more prone to chromosomal fusions than non-amplified lines ( P = 9.1 × 10 −6 ) (Figure 1 B), and ovarian CCNE1 -amplified cell lines showed the same trend compared with non-amplified cell lines ( P = 0.032) (Figur e 1 C). Furthermor e, these fusions ar e mor e likely to occur at cyclin E1-induced fragile sites ( Supplementary Figure S1A,B) ( 28 ). We also observed that CCNE1 -amplified cells tend to have higher levels of aneuploidy compared with non-amplified cell lines across tumor types ( P = 2.2 × 10 −9 ) (Figure 1 D), and in ovarian cancer cell lines specifically ( P = 0.0044) (Figure 1 E). This observation is consistent with the role of cyclin E1 in endoreplication during de v elopment (29)(30)(31). CCNE1 -amplified cells, howe v er, do not have higher mutational burden compared with nonamplified cells (Supplementary Figure S1C, D). Taken together, these data support that CCNE1 -amplified cell lines may be encountering barriers to replication in CCNE1induced fragile sites, leading to chromosomal fusion e v ents.

CCNE1 -amplified cells r equir e CDK2 f or CHK1 signaling, and HR for replication restart
To choose model cell lines to interrogate the relationship between CCNE1 amplification and HR, we measured protein le v els of key cell cycle regulators in a panel of HR-proficient CCNE1 -amplified and CCNE1 non-amplified cells (Figure 2 A, Supplementary Table S2). We chose OVCAR-3, FUO V1 and CO V318 to r epr esent ovarian cell lines with amplification of CCNE1 , and COV644 to r epr esent ovarian cell lines with two copies of CCNE1 . We also included a diploid, immortalized cell line, hTERT-RPE1, to interrogate a non-cancerous cell line with an intact cell cycle ( 32 ), and A549, a cell line previously reported to be dependent on CDK1 for HR (Supplementary Table S2) ( 19 ). As expected, CCNE1 -amplified cell lines had increased le v els of cy clin E1 protein (Figure 2 A), consistent with patient data from    Figure S2A). CCNE1 -amplified cell lines also had markers of acti v e CDK2 (ele vated phosphoryla tion a t T160, eleva ted le v els of cy clin A2, low p21) (Figure 2 A). Notably, CCNE1 -amplified cell lines also had high le v els of p16, a negati v e regulator of cy clin-dependent kinase 4 / 6 (CDK4 / 6) ( 33 ), and elevated levels of pCDK1 Y15, an inhibitory phosphorylation site on CDK1 (Figure 2 A) ( 34 ). The protein le v els of these markers indicate that CDK2 may be the predominantly acti v e CDK in this setting.
Because the response to replication fork stalling is controlled mainly by the CDK2-A TRIP-A TR-CHK1 axis ( 35 ), we utilized a selecti v e CDK2 inhibitor, BLU1851 (Supplementary Figure S2B), to interrogate this signaling cascade in CCNE1 -amplified cells. BLU1851 is a potent and specific CDK2 inhibitor with limited off-target activity against the entire kinome, including other related CDK family members, such as CDK1 (Figure 2 B, Supplementary Figure  S2C-H, Tables S3A-C) ( 36 ). To keep concentrations in a selecti v e range ov er the key off-target CDK1 for our mechanistic experiments, we tested BLU1851 in cellular phosphorylation assays to measure CDK2 and CDK1 activity. We measured phosphorylation of Rb in OVCAR-3 as a CDK2 readout ( 37 ), and phosphorylation of lamin A / C as a CDK1 readout ( 38 ) and found that there is a large window between CDK2 and CDK1 activity (Supplementary Figure S2F,G). Lastly, because there was some activity on CDK4 in the enzyme assay (Supplementary Figure  S2D, Table S3A), we tested the anti-proliferati v e effect of BLU1851 on a CDK4-dependent cell line, MCF-7, and calculated an average IC 50 > 1 uM, suggesting BLU1851 does not potently target CDK4 (Supplementary Figure S2H, Table S3C). Based on these data, we chose CDK2-selecti v e concentrations of BLU1851 in our studies.
Gi v en the ov erall chromosomal instability and mar kers of hyperacti v e CDK2 observ ed in CCNE1 -amplified cells, we hypothesized that these cells may be especially reliant on CDK2-CHK1 signaling in response to stalled or collapsed replication forks. We therefore measured CHK1 activation via S317 phosphorylation in both OVCAR-3 and COV644 cells after 2 and 24 h of hydroxyurea (HU) treatment to stall or collapse replication for ks, respecti v ely, in the presence or absence of BLU1851, at a concentration below the proliferati v e half-maximal inhibitory concentration (IC 50 ) in OVCAR-3 (Supplementary Table S3C) to limit the effects of the compound on the cell cycle. While CHK1 signaling was expectedly activated in both cell lines following HU treatment, CDK2 inhibition suppressed pCHK1 S317 to a greater extent in OVCAR-3 cells compared with COV644 cells (Figure 2 C, Supplementary Figure S2I). BLU1851 treatment also inhibited pCHK1 S317 in FUOV1 and COV318 to a greater extent than in hTERT-RPE1 or A549 cells (Supplementary Figure S2J). These data support a more pr ominent r ole for CDK2 in replication fork maintenance via CHK1 in CCNE1 -amplified cells compared with non-amplified cells.
As an avenue f or f ork restart or repair, cells can use HRmediated mechanisms such as DSB repair after nucleolytic cleavage of a r egr essed fork, template switching, and gap repair, or they can utilize alternati v e, more mutagenic pathways such as translesion synthesis (TLS) ( 39 ). To first vali-da te tha t we could pre v ent proliferati v e recov ery from replication fork collapse via HU, we treated OVCAR-3 cells with the CHK1 inhibitor, rabusertib, at a concentration that would not inhibit proliferation on its own followed by a 24h pulse of HU ( 40 ) (Supplementary Figure S2K). Indeed, CHK1 inhibition suppressed the ability of OVCAR-3 cells to proliferate following HU treatment ( Supplementary Figure S2K). We next utilized this assay to measure the ability of OVCAR-3 and COV644 cells to resume proliferation after HU-induced replication fork collapse by using small interfering RNA to knockdown the HR factor RAD51, or the TLS factor, REV1. COV644 cells were overall less efficient at recovering from HU, while OVCAR-3 cells were able to resume proliferation and reach the confluence of vehicle-tr eated controls (Figur e 2 E). Howe v er, when we depleted RAD51 in OVCAR-3 cells, we observed that cells were less efficient at proliferating following HU treatment compared with non-targeting control, while REV1 depletion had no significant effect (Figure 2 D, E, Supplementary Figure S2L). In contrast, neither RAD51 nor REV1 depletion in COV644 cells worsened the recovery from HU compared with non-targeting control, albeit with less efficient knockdown compared to OVCAR-3 (Supplementary Figure S2M,N). These data support the hypothesis that CCNE1 -amplified cells rely, at least in part, on HRmediated mechanisms to repair collapsed replication forks, while non-amplified cells may utilize multiple repair pathways to compensate for the loss of one pathway.
Since CCNE1 -ov ere xpressing cells incur endo genousl y higher rates of DNA damage through accumulation of DSBs ( 10 ), and RAD51 knockdown impaired the ability of OVCAR-3 cells to recover from induced replication fork collapse, we reasoned that OVCAR-3 cells would be more sensiti v e to inhibitors that disrupt the HR pathway compared with COV644 cells if they do indeed utilize HR to comba t replica tion stress. To test this, we treated cells with mirin, which inhibits the e xonuclease acti vity of MRE11, streptonigrin, which inhibits RAD54 ATPase activity, and JH-RE-06, a REV1-REV7 pr otein-pr otein interaction inhibitor, to model inhibition of early-stage resection, later stage HR, and TLS, respecti v ely (41)(42)(43). OVCAR-3 cells wer e mor e sensiti v e to both mirin and streptonigrin compared with COV644 cells, while COV644 cells were more sensiti v e to JH-RE-06 (Figure 2 F). These data suggest that OVCAR-3 cells r equir e HR, but not TLS, for proliferation to a greater extent than COV644 cells.

Cyclin E1 is present at the site of stalled replication forks in CCNE1 -amplified cells
Since CCNE1-amplified cells are dependent on CDK2 for replication fork stall signaling, we measured the physical presence of CDK2 and its canonical cyclin partners, cyclin E1 and cyclin A2, at the site of stalled forks in two CCNE1 -amplified lines, OVCAR-3 and FUOV1, and two non-amplified lines, COV318 and hTERT-RPE1. We used a proximity ligation assay ( 44 ) to detect the presence of cyclin E1, cyclin A2 and CDK2 at 5 -ethylene-2 -deoxyuridine (EdU) labeled nascent DNA stalled by HU (Figure 3 A) ( 45 ). Surprisingly, O VCAR-3 and FUO V1 cells not only had significantly more cyclin E1 at the site of stalled replication forks compared with COV644 cells, but also significantly more CDK2 and cyclin A2 (Figure 3 B , C , Supplementary Figures S3A, B). The physical proximity of CDK2 along with its canonical cyclin partners suggests that catal yticall y acti v e CDK2 has a role directly at the site of the stalled fork. Additionally, the detection of cyclin E1 at stalled forks in CCNE1 -amplified cell lines suggests that cyclin E1 could be participating in non-canonical replication activities outside of its known functions in pr e-r eplication comple x assemb ly ( 46 ). While COV644 cells had less CDK2 and cyclins present at stalled forks compared with OVCAR-3 and FUOV1 cells, hTERT-RPE1 did not display significant differences in cyclin A2 levels, but did have significantly fewer instances of CDK2 recruitment and trended toward less cyclin E1 levels (Figure 3 B, Supplementary Figure  S3A). Overall, these data suggest that CDK2 is acting physically at the site of stalled replication forks in the CCNE1amplified setting.

CDK2 regulates homologous recombination in CCNE1amplified o v arian cells
Because CCNE1 -amplified cells r equir e CDK2 for stalled fork signaling via CHK1 (Figure 2 C, Supplementary Figure  S2H), and may restart forks through HR (Figure 2 E), we hypothesized that CDK2 may be the e xclusi v e kinase regulating HR in tumors with this genetic background. To test this, we engineered stable cell lines e xpressing doxy cy clineinducible CDK2 and CDK1 short hairpin RN A (shRN A) in a panel of CCNE1 -amplified and non-amplified cell lines. After knockdown of CDK2 or CDK1 in each line, we treated cells acutely with the topoisomerase II inhibitor, etoposide, to induce DSBs ( 47 ). We confirmed induction of DNA damage by pH2AX S139, and examined HR signaling via CtIP bandshift and pRPA S4 / 8 le v els as a measure of DNA end resection ( 48 , 49 ) (Figure 4 Figures S4B-F). Consistent with the data from inducible CDK2 knockdown, the CDK2 inhibitor BLU1851 suppressed HR signaling as measured by the CtIP bandshift and pRPA S4 / 8 in CCNE1 -amplified cell lines in a dosedependent manner but had little effect in non-amplified cell lines (Figure 4 B, Supplementary Figure S4G, H). While the selecti v e CDK1 inhibitor, RO-3306 ( 50 ), had only a small effect in some cell lines, the CDK1 / 2 dual inhibitor, roscovitine ( 51 ), suppressed HR signaling in most cell lines tested (Figure 4 B, Supplementary Figure S4G, H). Taken together, these data suggest that CDK2 may be regulating HR nonredundantly in CCNE1 -amplified cells.
To test if CDK2 inhibition can inhibit HR in CCNE1amplified cells, we employed a CRISPR / Cas9-based recombination assay to induce a targeted DSB upstream of the beta-actin stop codon and provided a donor template with an inserted 3X-FLAG reporter ( 52 ) (Figure 4 C). As expected from the signaling data (Figure 4 B, Supplementary Figur e S4H), r ecombination was suppr essed in OVCAR-3 and FUOV1 cells but not in COV644 or hTERT-RPE1 cells after treatment with BLU1851 (Figure 4 D, E, Supplementary Figure S4I). Treatment with roscovitine, in contrast, inhibited recombination in all cell lines (Figure 4 D, E, Supplementary Figure S4I). These data suggest that, in the CCNE1 -amplified setting, CDK2 is uniquely r equir ed for recombination, while CDK1 and CDK2 may have redundant roles in non-amplified cells.
Because repair pathway choice is in part dictated by cell cycle phase ( 53 ), and CDK2 inhibition arrests cells at the G1 / S boundary in CCNE1 -amplified cells (Supplementary Figure S4J), we tested if the suppression of HR signaling after CDK2 inhibition in CCNE1 -amplified cells was a consequence of cell cycle arrest or if CDK2 was directly acting in the HR pathway. We compared cells arrested at G1 / S with double thymidine block or with BLU1851 treatment. While both methods arrested cells in G1 as measured by flow cytometry (Supplementary Figure S4K), only treatment with the CDK2 inhibitor suppressed HR signaling after etoposide treatment (Supplementary Figure S4L). These da ta support tha t CDK2 is functioning in HR directly and the observed effects are not simply a consequence of cell cycle arrest.

CDK2 inhibition sensitizes CCNE1 -amplifed cells to DNA damaging agents in vitro and in vivo
We have shown that CDK2 loss or inhibition suppresses HR signaling and recombination, and thus reasoned that combining CDK2 inhibition with a chemotherapy that induces DSBs, such as etoposide, should be synergistic. We tested a dose matrix of BLU1851 and etoposide in OVCAR-3 and COV644 cells in vitro and observed that the combination was synergistic in OVCAR-3 cells (highest single agent [HSA] = 13.97), but not in COV644 cells (HSA = 5.03) (Figure 5 A). Notably, the strongest le v els of synergy in OVCAR-3 cells were observed at low concentrations of both agents, below the proliferati v e IC 50 . These same doses in COV644 cells trend toward antagonism. The combination effect at low doses in OVCAR-3 cells suggests that the underlying mechanism is based on the DNA damage function, rather than the proliferati v e function, of CDK2. These data support the hypothesis that CDK2 regulates HR in CCNE1amplified cells.
To test if a CDK2 inhibitor in combination with etoposide is anti-tumorigenic in vivo , we used the OVCAR-3 xenograft, a model previously used to validate CDK2 as an oncogenic dri v er in CCNE1 -amplified ovarian cancer ( 54 ). In these studies, we used BLU2256, a second selecti v e CDK2 inhibitor that suppresses HR signaling and recombination in CCNE1 -amplified cells. BLU2256 is structurally related to BLU1851 and showed similar in vitro phenotypes, but with superior pharmacokinetics, making it more suitable than BLU1851 for in vivo studies (Supplementary Figures S5A-J, Tables S4A-C) ( 36 ). BLU2256 demonstrated dose-dependent tumor growth inhibition in OVCAR-3 xenografts without a change in body weight (Figure 5 B , C , Supplementary Figure S5K). We determined on-target activity of this agent by measuring the inhibition of RB1 phosphorylation, which was dose and   Figures S5L,M). Based on the dose-titra tion da ta, we determined tha t a 3 mg / kg QD dose was sub-efficacious with minor tumor growth inhibition, while 5 mg / kg twice daily (BID) was an efficacious dose, inducing tumor stasis. We tested both doses of BLU2256 in combination with a low-dose of etoposide (1.25 mg / kg) and observed there is only a benefit of combina tion trea tment when CDK2 is inhibited sub-optimally at 3 mg / kg QD, consistent with the in vitro data ( Figure 5 D, E, Supplementary Figure S5N). Because HR impairment is synthetically lethal with PARP inhibition ( 2 , 3 ) we reasoned that a CDK2 inhibitor, in combination with a PARP inhibitor should show greater anti-tumor activity compared to single agent. To test this, we treated mice harboring the OVCAR-3 xenograft model with BLU2256 at 3 mg / kg QD, olaparib at 50 mg / kg QD, or the combination. We observed that the combination of BLU2256 and olaparib had better anti-tumor activity compared to the single agent tr eatments (Figur e 5 F,G, Supplementary Figure S5O). These data support a mechanism by which CDK2 inhibition suppresses HR in CCNE1amplfied cells and induces an anti-tumorigenic phenotype when in combination with DNA damaging agents.

DISCUSSION
This study provides a mechanism to explain the mutual exclusivity between CCNE1 amplification and HRD in HG-SOC. We propose that CCNE1 -amplified ovarian cancer cells rely on HR to resume replication after fork collapse. We also measured high le v els of CDK2, cyclin E1, and cyclin A2 protein at the site of stalled replication forks in CCNE1 -amplified cells which may indicate direct CDK2 ca talytic activity a t stalled forks. Lastly, we demonstra te that CDK2 is non-redundantly regulating stalled / collapsed replication fork signaling via CHK1 and HR following induced DNA damage in CCNE1 -amplified cells. Inhibition of CDK2 in combination with etoposide showed better efficacy compared with a single agent at low doses in vitro and in vivo . This work suggests CDK2 inhibitors in combination with DNA damaging agents may have added clinical benefit.
While previous studies have noted the mutual exclusivity between CCNE1 -amplification and HRD ( 4 ), the molecular process governing this relationship has not been adequately detailed. Our proposed mechanism that CCNE1amplified cells utilize HR to combat r eplication str ess is consistent with reports that HR genes ar e upr egulated following CCNE1 ov ere xpression in fallopian tube secretory epithelial cells, the presumed cell of origin for HGSOC ( 55 ). A similar mechanism may be apparent in cancer cell lines that grow despite the constant replication stress imposed by high cyclin E1 levels. The frequency of replication fork collapse in CCNE1 ov ere xpressing human fibroblast cells reveals unique fragile sites in the genome, many of which are instability hot spots in cancer and are prone to rearrangement ( 28 ). Consistent with this finding, our GCR analysis re v ealed that CCNE1 -amplified cells ar e mor e likely to have chromosomal fusion events overall, and these fusion e v ents are more likely to occur in CCNE1 -induced fragile sites. The higher le v els of aneuploidy observ ed in CCNE1amplified cells are also consistent with the hypothesis that r eplication str ess imposed by CCNE1 -le v els induces GCR. In contrast, we did not observe a higher mutational burden in CCNE1 -amplified cells, which would be associated with more err or-pr one pr ocesses, such as NHEJ or replication restart through TLS. These data support a hypothesis that CCNE1 -amplified cells have barriers to replication and may be using HR as a method to resume replication at the site of replication fork stall / collapse.
The role of CDK2 in this process has been less clear, as a recent report suggested that unrestrained CDK2 activity can promote excessive fork degradation ( 56 ), despite known roles in maintaining genome integrity ( 57 , 58 ). A major limitation and potential confounding factor in past studies is the use of non-selecti v e kinase inhibitors to interrogate CDK functions, or the use of a CDK2 analog-sensiti v e mutant that has impaired cyclin binding and thus low kinase activity ( 59 ). Here, we utilized specific and potent CDK2 inhibitors with selectivity not only over other CDK family members but also the entire kinome, to more precisely assess how CDK2 is promoting repair signaling in CCNE1amplified cells. Our study suggests CDK2 is r equir ed in CCNE1 -amplified cells both for replication fork signaling, as well as HR but is dispensable in non-amplified cells. To scores in Synergy Finder. A score ← 10 is indicati v e of antagonism, a score between -10 and 10 is indicati v e of additi vity, and a score > 10 is indicati v e of synergy. Data are representati v e of at least four independent experiments. ( B ) Tumor volume over time in mice harboring OVCAR-3 xenografts treated with vehicle or BLU2256 at indicated doses QD for 3 weeks. N = 8 / treatment group. A two-way ANOVA repeated measures (RM) was performed on day 21 to compare the tumor volume between treatment groups and vehicle groups. Comparisons between groups were carried out with Tukey multiple comparison test. P < 0.05 was considered to be statistically significant. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ( C ) Tumor volume at day 21 shown in (B). A one-way ANOVA RM was performed to compare the tumor volume between treatment groups and vehicle groups. Comparisons betw een groups w ere carried out with Tukey multiple comparison test. P < 0.05 was considered to be statistically significant. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ( D ) Tumor volume over time in mice harboring OVCAR-3 xenografts treated with vehicle, BLU2256 at indicated doses, Etoposide at 1.25 mg / kg (3 days on, 4 days off). N = 8 / treatment group. A two-way ANOVA RM was performed on day 28 to compare the tumor volume between treatment groups and vehicle groups. Comparisons between groups were carried out with Tukey multiple comparison test. P < 0.05 was considered to be statistically significant. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ( E ) Tumor volume at day 28 shown in (D). A one-way ANOVA RM was performed to compare the tumor volume between treatment groups and vehicle groups. Comparisons between groups were carried out with Tukey multiple comparison test. P < 0.05 was considered to be statistically significant. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. ( F ) Tumor volume over time in mice harboring OVCAR-3 xenografts treated with vehicle, BLU2256 at indicated doses, olaparib at 50 mg / kg QD. N = 8-10 / treatment group. A two-way ANOVA RM was performed on day 24 to compare the tumor volume between treatment groups and vehicle groups. Comparisons between groups were carried out with Tukey multiple comparison test. P < 0.05 was considered to be statistically significant. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. (G) Tumor volume at day 24 shown in (F). A one-way ANOVA RM was performed to compare the tumor volume between treatment groups and vehicle groups. Comparisons between groups were carried out with Tukey multiple comparison test. P < 0.05 was considered to be statistically significant. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001. our knowledge, this is the first report demonstrating exclusi v e dependence on CDK2 for DNA repair.
Prior to this study, neither cyclin A2 nor cyclin E1 had been detected at the site of stalled replication forks using methods such as isolation of proteins on nascent DNA (iPOND) ( 60 ), potentially because this technique is not suited for transient or low abundance interactions. The data pr esented her e indicate acti v e CDK2 is directly associating with stalled replication forks, especially in the CCNE1amplified setting. In contrast, CDK2 does not appear to be a prime actor in replication fork maintenance in CCNE1 non-amplified cell lines. The physical presence of CDK2, cyclin A2, and cyclin E1 at stalled replication forks in CCNE1amplified cells may indicate these complex es ar e functioning to promote repair and restoration of the fork. Determining the precise function of CDK2-cyclin E1 or CDK2-cyclin A2 at the site of stalled forks in CCNE1 -amplified cells is an area of future research.
Loss of CDK2 function can typically be compensated for by other CDK family members, especially by CDK1 ( 61 , 62 ). Indeed, in previous reports, CDK1 was identified as essential for HR and synergized with DNA dama ging a gents, while CDK2 was dispensable ( 19 , 24 ). The present study shows that CDK2 is essential for HR in the CCNE1 -ampified context and selective inhibition of CDK2 a t sub-ef ficacious doses can sensitize cells to DNA damaging agents. CDK2 is an attracti v e therapeutic target as a single agent in CCNE1 -amplified cancers as cell lines and xenograft models harboring the amplification fail to proliferate after knockdown or catalytic inhibition of CDK2 ( 54 ). W hen trea ted in combina tion with etoposide or olaparib, we demonstrated added anti-tumor activity when CDK2 inhibition was sub-efficacious. Because PARP inhibitors have had a major clinical impact in HR-deficient tumors, inducing an HRD phenotype to re v eal synthetic lethality with a PARP inhibitor is attracti v e ( 2 , 3 ). These findings will also be important for clinical applications if patients receiving CDK2 inhibitors need to dose reduce, or to limit potential drug-drug interactions when treated in combination. These data support a CDK2-dri v en HR mechanism in CCNE1amplified HGSOC.
Our data suggest a model in which CCNE1 -amplified cell lines rely on HR to repair collapsed replication forks. In addition to its role as a cell cycle regulator, CDK2 non-redundantly coordinates HR in this genetic context. While CCNE1 -amplified cells have engaged HR as a survival mechanism to comba t replica tion stress, inhibiting this process leaves them vulnerable to synthetic lethality with chemotherapies. As CDK2 inhibitors enter the clinic for HGSOC (clinicaltrials.gov: NCT04553133, NCT05252416), rational drug combinations could open additional clinical avenues and improve patient outcome.

DA T A A V AILABILITY
The data underlying this article will be shared on reasonable request to the corresponding author.